The familial amyloid polyneuropathies (FAPs) are systemic amylid syndromes which are inherited in an autosomal dominant fashion (Glenner, G. G. et al., The Metabolic Basis of Inherited Disease pp 1308-1339, McGraw-Hill, New York 1978). They are characterized by the deposition of fibrils containing the plasma protein prealbumin (transthyretin) (Costam, P. P. et al., Proc. Natl. Acad. Sci., USA (1978) 75:4499; Benson, M. D., J. Clin. Invest. (1981); 67: 1035; and Skinner, M. et al., Biochem. Biophys. Res. Comm. (1981) 99: 1326.). Prealbumin is a 55 K Mr protein, composed of four identical subunits, which is involved in thyroid hormone and vitamin A transport (Blake, C.C.F., Proc. R. Soc. Lond. (1981) B211: 413.) Recently prealbumin isolated from the amyloid deposits of FAP patients has been sequenced and shown to have various single amino acid substitutions not found in the circulating prealbumin of normal individuals. One FAP associated prealbumin variant has a methionine for valine substitution at amino acid 30 and is found in kinships of Portuguese (Saraiva, M. J. M. et al., J. Clin. Invest (1984) 74: 104), Japanese (Tawara, S. et al., Biochem. Biophys. Res. Comm. (1983) 116: 880) and Swedish (Dwulet, et al., Proc. Natl. Acad. Sci., USA (1984) 81: 694; and Whitehead, A. S. et al., Mol. Biol. Med. (1985) Vol. 7 in press) ancestry type 1 (FAP). This prealbumin variant is present in the plasma of some kinships afflicted with type 1 FAP and it has been proposed that peptide mapping would provide a definitive diagnostic test for this form of the disease (Saraiva, M. J. M. et al., supra; Benson, M. D. et al., J. Clin. Invest. (1985) 75: 71.) Another type 1 FAP associated prealbumin variant has been identified by Whitehead et al., Mol. Biol. Med. supra. The variant prealbumin allele associated with type 2 FAP has a serine for isoleucine substitution at position 84 (Wallace et al., Clin. Res. (1985) 33: 592A). Variant prealbumin alleles have also been implicated in Alzheimers Disease (Shirahama, T. et al., Am. J. Pathol. (1982) 107: 41; A. S. Cohen and J. Sipe, NIH Grant No. P50-AG/NH05141 original submitted 3/19/84, revised 11/07/84).
There are three basic methods currently in use for detecting variant protein alleles: detecting the altered protein molecule itself; determining the nucleotide sequence of the coding regions of the variant gene; and detecting mutations that affect Southern blot hydridization patterns arising from either insertions or deletions in the gene or point mutations which either create or destroy restriction endonuclease recognition sequences.
The method described by Benson et al. for detecting carriers of the Met 30 human prealbumin variant falls into the first of the basic methods described. (Benson, M. D. et al., supra.) Since normal prealbumin has only one methionine (position 13), treatment with cyanogen bromide (CNBr), which cleaves only at methionines, results in two peptides. CNBr treatment of the Met 30 variant gives three peptides. The extra peptide is detected by high performance liquid chromatography (HPLC) or sequential Edman degradation.
Nakazato et al. report a radioimmunossay for the Met 30 variant of human prealbumin based on a nonapeptide (position 22-30) of the prealbumin variant. (Nakagato, M. et al., The Lancet (1985) 1: 99).
The second approach described above to determine the DNA structure of the mutant gene is a long and labor intensive process requiring cloning of normal and variant alleles. As such there is not much interest in this approach.
In order to detect variant prealbumin alleles by the third approach described above, use of restriction endonuclease based assays, it is necessary to clone and sequence cDNA for the prealbumin variant of interest. Recent advances in biochemistry and in recombinant DNA technology have made it possible to clone DNA and to achieve the controlled synthesis of specific proteins using the technique of molecular cloning. Molecular cloning involves isolating and purifying a nucleotide sequence coding for a particular protein, inserting the sequence into a plasmid or other cloning vehicle and transferring the cloned gene to a suitable cell for amplification and/or expression of the protein. Maniatis, T. et al.--Molecular Cloning--A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, 1982. The protein can then be isolated and purified by conventional techniques.
Restriction endonucleases are enzymes, isolated primarily from prokaryotes, which recognize specific nucleotide sequences within a DNA molecule (Maniatis et al., supra, at pp 98-106). Generally, restriction endonucleases may be classified into three groups. Type-I and type-III enzymes carry methylase and an ATP-requiring cleavage activity in the same protein. Both types of this enzyme recognize unmethylated sequences in a substrate, but type-I enzymes cleave randomly, while type-III enzymes cleave at specific sites. Type-II restriction enzymes consist of a separate restriction endonuclease and modification methylase. A large number of type-II enzymes have been isolated (Roberts, R. J. Nuc. Acids Res. (1982) 10:R117), many of which are useful in molecular cloning. These enzymes cut DNA within or near a particular recognition sequence, which typically is from four to six nucleotides in length. In general, different restriction enzymes recognize different target nucleotide sequences. However, there are several enzymes isolated from different sources which recognize the same target sequences. These enzymes are known as isochizomers. Most restriction enzymes recognize either tetranucleotide, pentanucleotide or hexanucleotide sequences; see Roberts, R. J., Nuc. Acids Res. (1983) 11:R135.
A restriction endonuclease has been employed in an assay for detecting the sickle cell allele (beta.sup.S gene) (Wilson et al., U.S. Pat. No. 4,395,486). Wilson et al. analyzed amniotic fluid by isolating DNA therefrom, digesting the DNA with the restriction enzyme Dde I, which recognizes the nucleotide base sequence CTNAG, and separating the DNA fragments following cleavage. Using standard hybridization probe detection methods and a specific probe for the human beta-globin gene, the presence of an approximately 376 base pair fragment and the absence of an approximately 175 base pair fragment in the analyte was stated to be indicative of the presence of the sickle cell genotype.
It would be advantageous to develop diagnostic tests for medical conditions associated with variant prealbumin alleles using hybridization based assays. Moreover, it is desirable to have a recombinant source of human prealbumin that can supply normal prealbumin protein substantially free of other proteins of human origin. Thus, there is much interest in cloning cDNA for human prealbumin in quantities and purity sufficient for use in clinical applications.